We propose to use time-resolved fluorescence resonance energy transfer (FRET) to recover conformational and/or molecular distributions of donors and acceptors in macromolecular systems. Both time-domain (TD) and frequency-domain (FD) methods will be utilized. We will extend our present capabilities with linked donor (D) -acceptor (A) pairs to the more complex case of un-linked pairs, with and without D-to-A diffusion. Such models are of interest for the study of non-random lipid distributions in membranes and around membrane-bound proteins, and clustering of ions around polyelectrolytes. The theory and measurements will be extended to include molecular geometric factors, such as the excluded volume around protein-bound or membrane-bound donors, or the distance of closest approach of ions to DNA. Other goals of the upcoming grant period include: l. Chemical synthesis of conjugatable donors which display long lifetimes from 200 to 4,000 ns. Such probes may enable detection of domain-to- domain motions in multi-domain proteins, such as troponin C, creatine kinase, or protein kinase C, or immunoglobulins, and allow measurement of lateral diffusion in bilayers on a previously inaccessible timescale. Collaborations have been established for these protein and membrane systems. 2. Application of our multiple methods for enhanced resolution of distance distribution to resolve one-step (cooperative) versus continuous alpha- helix formation in helix-forming peptides and melittin. These D and A- labeled model systems will be synthesized. 3. Develop the use of the acceptor decay kinetics, and/or selective two- photon excitation of the donors, to recover distance distributions. 4. Evaluate the use of distance distribution and site-to-site diffusion measurements of linked D-A pairs in bilayers as an indicator of membrane order and dynamics. We will use both short (5 ns) and long (60 ns) lived donors. 5. Measurement of tyrosine-to-tryptophan energy transfer, with development of software to account for spectral overlap at all practical observation wavelengths. 6. All the above topics require development of appropriate software, for simultaneous (global) analysis of TD and FD data obtained for the same samples.